The present invention relates to methods of analyzing DNA replication, and more particularly to the use of molecular combing to facilitate the detection and identification (i.e., mapping) of origins of replication and the measurement of the dynamic and structural relationships regulating DNA replication.
Control of DNA synthesis is essential for maintaining genome stability (1, 2, 3). In higher eukaryotes, genomic instability associated with a loss of replication control and aberrant DNA synthesis is a key feature of a variety of neoplasms and genetic diseases (4, 5, 28). In yeast, an altered pattern of DNA synthesis leads to genomic abnormalities including aneuploidies and translocations (6, 7). The efficient and accurate replication of the genome in eukaryotes is accomplished by the activation of multiple bidirectional origins of replication. The temporal and spatial pattern of activation, or replication program, varies according to the developmental stage (8). In X. laevis, for example, the duration of the period of DNA replication during the cell cycle depends upon replicon size, or the distribution of replication origins (9). However, the organization and distribution of replication origins throughout the eukaryotic genome is not well known, and consequently the regulation of the replication program in these cells is poorly understood (10). This is primarily due to a variety of technical and fundamental obstacles which make it difficult to study DNA replication at the genomic level (11). Though a number of techniques exist for studying DNA replication in both higher and lower eukaryotes, more rapid methods are needed for the quantitative analysis of the dynamics of genome duplication (11).